Our environment contains a multitude of microorganisms with which we are continuously interacting. These interactions can be beneficial, e.g. fermentations to produce wine, vinegar or antibiotics; neutral; or even harmful, as in the case of infectious diseases. The ubiquitous presence of these microorganisms, thus, creates a continuing need for the detection, identification and study of the presence and metabolic activity of such microorganisms.
While the science of microbiology has changed significantly in the last 25 years, many procedures for the detection, identification and analysis of the behavior of microorganisms are still time consuming. For example, in the area of antimicrobic susceptibility testing nearly half of all testing in hospitals in the United States still use the Bauer-Kirby Disc Method. This method uses the presence or absence of visible growth of the microorganisms to indicate the efficacy of an antimicrobic compound, and generally requires an 18 to 24 hour incubation period to allow for microorganism growth before a result can be obtained. A decrease in the time required to obtain such antimicrobic susceptibility information is needed.
Another popular method for antimicrobic susceptibility testing is the broth micro-dilution method, such as the Sceptor.RTM. System for identification and antimicrobic susceptibility testing of organisms (Becton Dickinson Diagnostic Instrumentation Systems, Sparks, Md.). The system uses a disposable plastic panel having a plurality of low volume cupulas (ca. 0.4 ml per cupula), each containing a different test compound or a different concentration of a test compound dried on the cupula surface. The organism to be tested is suspended in the desired testing medium, and aliquots are delivered to the individual cupulas of the test panel. The reagent dried on the panel dissolves in the sample, and the system is then incubated overnight (18 to 24 hrs.) to allow sufficient time for the organisms to interact with the reagent and for visible growth to appear. The panel is subsequently examined visually for the presence or absence of growth, thereby obtaining information on the susceptibility of the organism undergoing testing. Additional wells aid in identifying the organism. However, this test method suffers from the drawback of also requiring a long incubation period.
One approach to the reduction of the incubation time is to monitor metabolic activity of the microorganisms, rather than growth of colonies. Many approaches have been reported in the attempt to rapidly and accurately monitor such metabolic activity.
For example, apparatus utilizing light scattering optical means have been used to determine susceptibility by probing the change in size or number of microorganisms in the presence of various antimicrobic compounds. Commercial instruments utilizing these principles are exemplified by the Vitec System (BioMerieux Corp.). This system claims to yield information on antimicrobic susceptibility of microorganisms within 6 hours for many organism and drug combinations. Other combinations can require as long as 18 hours before the antimicrobic susceptibility of the organism can be determined by this machine.
Additionally, modifications of the Bauer-Kirby procedure have been developed which allow certain samples to be read in four to six hours. However, such a system is "destructive" in nature, requiring the spraying of a developing solution of a color forming dye onto the test plate. Re-incubation and reading at a later time is, thus, not possible and if the rapid technique fails, the experiment cannot be continued for a standard evaluation at a later time.
A bioluminescent method based on the quantity of ATP present in multiplying organisms has been described as yielding results of antimicrobic susceptibility testing in four and half hours for certain compositions (Wheat et al.). However, the procedure tends to be cumbersome and broad applicability has not been shown.
Other approaches have involved monitoring of microbial oxygen consumption by the measurement of pH and/or hemoglobin color change, or by the use of dyes such as triphenyl- tetrazolium chloride and resazurin, that change color in response to the total redox potential of the liquid test medium.
The monitoring of the consumption of dissolved oxygen by microorganisms, as a marker of their metabolism, has been studied for many years. For example, C. E. Clifton monitored the oxygen consumption of microorganisms over a period of several days using a Warburg flask in 1937. This method measured the change in oxygen concentration in a slow and cumbersome manner.
The "Clark" electrode, a newer electrochemical device, is also commonly used to measure dissolved oxygen. Unfortunately, the Clark electrode consumes oxygen during use (thereby reducing the oxygen available to the microorganisms) and the "standard" size electrode is typically used only to measure volumes of 100 mls or greater to prevent the electrode from interfering with the measurements.
A "miniature" Clark electrode has been described, but this electrode is a complicated multi-component part which must, also, be in contact with the solution to be measured. While an oxygen permeable membrane can be used to prevent the electrode components of the device from interacting with the constituents of the test solution, the oxygen must still equilibrate between the test solution and the measurement system and is consumed once it passes the membrane.
Optical systems which can yield oxygen concentration data, have been developed to overcome the shortcomings of the Clark electrode systems. The main advantage of such optical methods is that the instrumentation required to determine quantitative value does not itself make physical contact with the test solution. Optical techniques allowing both colorimetric and fluorometric analyses for oxygen to be carried out rapidly and reproducibly are known, and costs for such analyses are often quite low. For example, several luminescent techniques for the determination of oxygen have been described which are based on the ability of oxygen to quench the fluorescence or phosphorescence emissions of a variety of compounds. However, such methods have not been adapted to microbial monitoring.
Other systems have been described that provide information on the presence, identity and antimicrobic susceptibility of microorganisms in a period of eight hours or less. Wilkins and Stones in U.S. Pat. No. 4,200,493 disclose a system that uses electrodes and a high impedance potentiometer to determine the presence of microorganisms. In U.S. Pat. No. 3,907,646 Wilkins et al disclose an analytical method which utilizes the pressure changes in the headspace over a flask associated with microbial growth for the detection and surveillance of the organisms. U.S. Pat. No. 4,220,715 to Ahnell, discloses a system wherein the head space gas above a test sample is passed through an external oxygen detector for determination of the presence of microorganisms. Ahnell, in U.S. Pat. No. 4,152,213, discloses a system for analysis by monitoring the vacuum produced by growing organisms in a closed head space above a test sample. U.S. Pat. No. 4,116,775 to Charles et. al is an example of the use of optical means based on the increase in turbidity or optical density of a growing microbial culture for the detection and monitoring of bacterial growth. A combined electro-optical measurement of birefringence of a test solution containing microorganisms is described in EPO 0092958 (Lowe and Meltzer).
It becomes apparent that a wide variety of methods have been applied to the detection and the antibiotic susceptibility testing of microorganisms. Many of these methods can only yield useful data when monitored by instruments dedicated to this task. Thus there exists a need for a system which can allow determinations of the presence and behavior of microorganisms without the requirement of dedicated instrumentation. Further there exists a need for a system that will allow the determination of the effect of a compound such as an antibiotic on a sample of microorganisms in a short time that does not significantly alter the behavior of the microorganisms.